Scandella D, DeGraaf Mahoney S, Mattingly M, Roeder D, Timmons L, Fulcher C A
Department of Molecular Biology Development, Rorer Biotechnology, King of Prussia, PA 19406.
Proc Natl Acad Sci U S A. 1988 Aug;85(16):6152-6. doi: 10.1073/pnas.85.16.6152.
Epitopes for antibodies that inhibit factor VIII procoagulant protein were analyzed by deletion mapping of factor VIII protein fragments expressed in Escherichia coli. A human factor VIII cDNA clone was used to generate E. coli expression vectors encoding fragments containing the 80-kDa factor VIII light chain (A3, C1, and C2 domains) and the 44-kDa carboxyl-terminal half of the factor VIII heavy chain (A2 domain). A series of deletions of each fragment was constructed and tested by immunoblotting for the binding of alloantibody and autoantibody inhibitors. Analysis of derivatives of the 80-kDa fragment showed that six inhibitors recognized a major epitope(s) within the carboxyl-terminal 17.3 kDa of factor VIII. These inhibitors also recognized weaker epitopes nearby and one inhibitor recognized epitopes scattered throughout the 80-kDa fragment. Deletions within the heavy chain fragment revealed one epitope-containing region confined to the amino-terminal 18.3 kDa recognized by six inhibitors. Bacterially produced factor VIII fragments containing the major epitopes were capable of neutralizing inhibitors in vitro but fragments containing weaker or no epitopes did not. These data suggest a potential therapeutic use of factor VIII fragments for neutralization of inhibitor antibodies.
通过对在大肠杆菌中表达的凝血因子 VIII 促凝蛋白片段进行缺失作图，分析了抑制凝血因子 VIII 促凝蛋白的抗体的表位。使用人凝血因子 VIII cDNA 克隆构建大肠杆菌表达载体，这些载体编码包含 80 kDa 凝血因子 VIII 轻链（A3、C1 和 C2 结构域）以及 44 kDa 凝血因子 VIII 重链羧基末端一半（A2 结构域）的片段。构建每个片段的一系列缺失，并通过免疫印迹检测同种抗体和自身抗体抑制剂的结合情况。对 80 kDa 片段衍生物的分析表明，六种抑制剂识别凝血因子 VIII 羧基末端 17.3 kDa 内的一个主要表位。这些抑制剂还识别附近较弱的表位，一种抑制剂识别遍布 80 kDa 片段的表位。重链片段内的缺失揭示了一个包含表位的区域，该区域局限于被六种抑制剂识别的氨基末端 18.3 kDa。含有主要表位的细菌产生的凝血因子 VIII 片段能够在体外中和抑制剂，但含有较弱或无表位的片段则不能。这些数据表明凝血因子 VIII 片段在中和抑制剂抗体方面具有潜在的治疗用途。
